A phylogenic analysis of the heme-globin family of proteins indicates that the well-characterized proteins hemoglobin and myoglobin were antedated by neuroglobin, which existed already 800 million years ago (Hankeln et al., J Inorg Biochem 99:110-119, 2005; Brunori and Vallone, Cell Mol Life Sci 64:1259-1268, 2007). Neuroglobin (Ngb) sequences remained highly conserved throughout mammalian evolution, suggesting a strongly selected vital functionality (Burmester et al., IUBMB Life 56:703-707, 2004). This heme containing, monomeric, 16.9 kDa protein shares 21-25% sequence homology with myoglobin and hemoglobin. However, unlike myoglobin and hemoglobin, it possesses a bis-histidine six-coordinate heme geometry, such that the proximal and distal histidines in the heme pocket are directly bonded to the heme iron (both Fe+2 or Fe+3 oxidation states) (Dewilde et al., J Biol Chem 276:38949-38955, 2001). Indeed, at equilibrium the concentration of the five-coordinate neuroglobin is very low, reported from 0.1 up to 5% (Uzan et al., Biophys J 87:1196-1204, 2004). Binding of oxygen or other gas ligands, such as nitric oxide (NO) or carbon monoxide, to the heme iron occurs upon displacement of the 6th coordination bond with the distal histidine 64 residue (Capece et al., Proteins 75(4):885-894, 2009; Kriegl et al., Proc Natl Acad Sci USA 99:7992-7997, 2002). Despite this structural difference with myoglobin, neuroglobin displays comparable α-helix globin folding and high oxygen affinity (P50 about 1-2 mmHg at 20° C.) (Kiger et al., IUBMB Life 56:709-719, 2004; Giuffre et al., Biochem Biophys Res Commun 367:893-898, 2008). However, the low tissue concentration of neuroglobin and the rapid auto-oxidation of the oxygen bound species suggest neuroglobin has not evolved to store and supply oxygen, leading to a number of different hypotheses about the physiological function of this conserved heme-globin (Brunori and Vallone, Cell Mol Life Sci 64:1259-1268, 2007; Burmester and Hankeln, J Exp Biol 212:1423-1428, 2009).
Despite uncertainty about the molecular functionality of neuroglobin, expression of this protein produces cytoprotective effects in vitro and in vivo, limiting neuronal cell death during glucose deprivation and hypoxia and limiting the volume of brain infarction in stroke models (Greenberg et al., Curr Opin Pharmacol 8:20-24, 2008; Khan et al., Proc Natl Acad Sci USA 103:17944-17948, 2006; Wang et al., Stroke 39:1869-1874, 2008; Sun et al., Proc Natl Acad Sci USA 98:15306-15311, 2001). An understanding of the functionality of neuroglobin could provide a paradigm shift in both biology and therapeutics, because many heme proteins in plants, bacteria, invertebrates and vertebrates are both highly conserved and exist in equilibrium between dominant six-coordinate geometry and the lower frequency five-coordinate state. Examples of these six-coordinate heme-proteins include cytoglobin, cytochrome c, Drosophila melanogaster hemoglobin, and the plant hemoglobins (Weiland et al., J Am Chem Soc 126:11930-11935, 2004; Nadra et al., Proteins 71:695-705, 2008; Garrocho-Villegas et al., Gene 398:78-85, 2007).
Over the last five years, groups have examined the ability of deoxygenated hemoglobin and myoglobin to react with and reduce nitrite to NO (Huang et al., J Clin Invest 115:2099-2107, 2005; Shiva et al., Circ Res 100:654-661, 2007). It has been proposed that this reaction serves a function similar to the bacterial nitrite reductases, in which a coupled electron and proton transfer to nitrite generates NO.Fe+2+NO2−+H+→Fe+3+NO.+OH−  (equation 1)
In the heart, myoglobin can reduce nitrite to NO to regulate hypoxic mitochondrial respiration and enhance the cellular resilience to prolonged ischemia, analogous to the cytoprotective effects of neuroglobin (Shiva et al., Circ Res 100:654-661, 2007). Studies using the myoglobin knockout mouse have now confirmed that myoglobin is necessary for nitrite-dependent NO and cGMP generation in the heart, nitrite-dependent cytoprotection after ischemia/reperfusion and nitrite-dependent control of hypoxic cellular respiration (Hendgen-Cotta et al., Proc Natl Acad Sci USA 105:10256-10261, 2008). It is therefore apparent that both myoglobin and neuroglobin may have roles in limiting cell death after ischemia-reperfusion injury. Of relevance to neuroglobin, it has recently been discovered that the mitochondrial protein cytochrome c can reduce nitrite to NO more rapidly than either hemoglobin or myoglobin, but only when it assumes the five-coordinate conformation (Basu et al., J Biol Chem 283:32590-32597, 2008). This conformation only occurs during the interaction with anionic phospholipids or upon oxidation or nitration of protein residues, suggesting a post-translational tertiary structure regulation of nitrite reduction and NO generation.
Interestingly, human neuroglobin contains two surface cysteines (C46 and C55) that form a disulfide bridge upon oxidation (Hamdane et al., J Biol Chem 278:51713-51721, 2003). Disulfide bond formation is accompanied by a decrease in the distal histidine binding affinity to heme iron (KHis, has been shown to decrease from ˜3000 to 280, values calculated as kon/koff are dimensionless) (Hamdane et al., Micron 35:59-62, 2004). This in turn increases the sub-population of five-coordinate neuroglobin and increases the affinity for endogenous ligands such as oxygen (P50 shift from about 9 to 1 mmHg) (Hamdane et al., J Biol Chem 278:51713-51721, 2003). Nicolis et al. reported that the oxidized disulfide-bridged neuroglobin also exhibits a higher affinity for nitrite than the thiol reduced form (Nicolis et al., Biochem J 407:89-99, 2007).